Scientific background report Phytophthora-resistant potatoes

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Phytophthora-resistant potatoes

Phytophtora infestans causes ‘late blight’. In regions of potato cultivation with a temperate

climate, this is the single most dangerous disease. The disease costs farmers in Belgium about

55 million euros annually, and controlling it causes significant environmental pressure.

However, in the last few years a number of resistant varieties based on conventional plant

breeding techniques were introduced to the market, and work is being done on developing

genetically modified Phytophthora-resistant lines. The conventional resistant varieties are the

result of a complex plant breeding program lasting many years. Two of the available varieties –

Bionica and Toluca – only contain a single resistance gene. The third – Sarpo Mira – a mixture of

different genes providing adequate resistance. These conventional varieties are still used in

small numbers; almost only in organic farming. Bionica and Toluca are only used in the fresh

vegatable market. Sarpo Mira is used to make fries, amongst others.

Currently there are also genetically modified Phytophthora-resistant potatoes being developed.

In many cases several resistance genes are introduced at once. And this is then done directly

into potato varieties that have properties that are of interest to the industry. Such potatoes are

still not available on the market, but are tested at various field test locations. In all probability it

will take until 2013 before such potatoes are available on the market.

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About this report

In this report the VIB (‘Vlaams Instituut voor Biotechnologie’) provides an overview of the

scientific knowledge of potatoes that are resistant to Phytophthora infestans, also known as

‘late blight’. VIB is a scientific institution with 1200 employees, with research groups at UGent,

K.U. Leuven, Universiteit Antwerpen and Vrije Universiteit Brussel. The Flemish Government

entrusted the VIB with disseminating evidence-based information on biotechnology.

About the references

Added to every paragraph you can find the references to the publications from which

information was used. Sometimes you can fully download these publications; sometimes they

are not freely available. In the latter case you can ask the author or look up the publication in

the nearest university library.

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Phytophthora-resistant potatoes .............................................................................................................. 2

About this report....................................................................................................................................... 3

About the references ................................................................................................................................ 3

A flourishing potato processing sector is present in Belgium .................................................................. 5

Phytophthora infestans – ‘late blight’ – is the largest threat to potato cultivation ................................. 6

Phytophthora infestans affects both plant and tuber, and is highly contagious ...................................... 6

Over the years, Phytophthora infestans turned more aggressive ............................................................ 8

Phytophthora infestans causes huge costs ............................................................................................... 9

Environmental pressure caused by controlling Phytophthora infestans .................................................. 9

Ways to keep the disease pressure of Phytophthora low ........................................................................ 9

Growing resistant varieties is the best way to reduce disease pressure ................................................ 10

Acting fast is essential when disease occurs........................................................................................... 10

Improved resistance with conventional plant breeding techniques ...................................................... 10

Phytophthora easily overcomes single resistance genes ........................................................................ 13

Introducing resistance against Phytophthora infestans by means of genetic manipulation ................. 14

Genetic modification for resistance against Phytophthora infestans step-wise .................................... 14

Cultivation of conventional resistant varieties ....................................................................................... 17

Sustainable resistance ............................................................................................................................ 17

BASF’s Fortuna potato ............................................................................................................................ 18

The DuRPh cisgenic project ..................................................................................................................... 18

Cisgenesis is subject of regulatory discussions in the EU ....................................................................... 19

Genetically modified Phytophthora-resistant potatoes do not threaten the environment................... 19

Food safety of genetically modified Phytophthora-resistant potatoes .................................................. 20

Genetically modified Phytophthora-resistant potatoes not on the market for now ............................. 21

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Potatoes are globally cultivated and Belgium is no exception

Potato cultivation is important as a source of food and starch. Potatoes are cultivated in more

than 100 countries. In 2006 the total global was 315 million tonnes of potatoes. China and India

together produce more than 30% of all potatoes.

Originally, potatoes come from the South American Andes, a place where they have been

cultivated for over 7000 years. Explorers brought them back to Europe in 1565, where they

were kept in botanical gardens for a long period of time. After successive crop failures of

traditional grains, they were introduced as food crop and began spreading over the whole

world, starting from Europe. Potatoes grow best in temperate climates in which the frost-free

period is sufficiently long. At temperatures under 10 oC and over 25 oC growth of the tuber is

restrained.

Three types of growing potatoes can be distinguished: seed potatoes, consumption potatoes

and starch potatoes. Growing seed potatoes is used for multiplying planting material. The

Netherlands specialises in this field and exports planting material all over the world. Growing

potatoes for human consumption is most important. It provides the potatoes we all eat, from

potatoes for cooking to deep-frozen French fries, but also snacks like crisps. Starch potatoes

produce starch for industrial applications: for adhesives, textile, paper, construction materials,

etc. Starch potato varieties are specially selected for their suitability for producing starch for

industrial applications. Starch potatoes can be eaten, but they are not very tasty.

In Belgium, the potato acreage has grown seriously in the last years to almost 81.000 hectares

in 2010. This results in a growing annual potato production of more than 3 million tonnes.

Flanders, the Dutch-speaking part of Belgium, produces most of this, 55 to 60% of the total. In

Belgium, the vast majority of potato production consists of potatoes for consumption. The

remainder consists of seed potatoes. In Belgium no starch potatoes are grown.

www.potato2008.org

A flourishing potato processing sector is present in Belgium

In Belgium, annually about 3 million tonnes of potatoes are processed in a flourishing and

expanding potato processing sector. A large majority of companies in this sector are family

businesses, which however have a strong international orientation.

The fact that the amount of potatoes produced in Belgium is equal to the amount that is

processed in the country does not imply that all Belgium potatoes are processed within the

country's borders. An intensive trade exists with the countries surrounding Belgium, as a result

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of which part of the potatoes produced in Belgium are processed abroad and part of the

potatoes processed in Belgium are produced abroad.

Consumption potatoes are processed for a broad range of potato products in Belgium: from

(deep frozen) French fries to mush, from cubes to wafers and from crisps to potato flakes. In

contrast with the potato processing industry in some other countries, the potato processing

companies in Belgium hardly have their own brands. Lutosa is one of the few exceptions. Most

of the companies produce for other (house) brands.

www.belgapom.be

Phytophthora infestans – ‘late blight’ – is the largest threat to potato

cultivation

Potato cultivation suffers from various diseases and pests, but ‘late blight’ forms the biggest

threat in regions with a temperate climate. Other threats are nematodes (Globodera pallida,

Globodera rostochiensis and Meloidogyne spp), and some bacterial diseases such as brown rot

(Ralstonia solanacearum), ring rot (Clavibacter michiganensis) and blackleg (Dyckeya spp). Late

blight is caused by the oomycete or water mould Phytophthora infestans, which is a fungus-like

organism. Late blight is best known for the Great Famine it caused in Ireland around 1845. In a

number of years the disease caused big potato crop failures in Ireland and proved to be a

turning point in Irish history. About a million Irishmen died as a result of the famine and about

the same number migrated, mostly to the U.S. to start a new life there. Phytophthora infestans

emerged in Central America and crossed the Atlantic Ocean to Europe from there in 1843. The

Great Famine took place only two years after.

The susceptibility of potatoes for Phytophthora infestans differs from variety to variety, but

only very few varieties are characterized by complete resistance. The Bintje variety, which

today still makes up between 40 to 50% of Flemish potato production, is highly susceptible to

late blight. Potato farmers in Flanders spray their crops 10 to 15 times each year with agents

that involve significant pressure to the environment. The number of times a crop is sprayed also

strongly depends on weather conditions.

Phytophthora infestans affects both plant and tuber, and is highly contagious

Phytophthora infestans affects leaves, stems and tubers. When a plant is affected, lesions

(spots on the leaves) occur on the leaves. How these spots look, depend on weather conditions.

Usually one can see spots with a 1 to 2 cm diameter, which slowly turn dark brown. Initially, the

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brown spots are lined by a yellow ring. In damp weather conditions, the underside of the leaf

will show a white fluffy mould. Under conditions that are favourable to the mould, affected

leaves will ultimately shrivel up completely. The stems display large elongated lesions with a

brown colour, reaching around the entire stem. The tubers are affected via buds or cracks in

the skin; contamination can occur both during growth, harvesting and storage. Damage

manifests itself as bluish spots becoming apparent from behind the skin, that turn rusty brown

over time. Often, infestation of the tuber leads to dry rot or wet rot (bacterial infection) and the

wet rot may then be passed on to unaffected tubers. In this way a limited number of tubers

affected by Phytophthora infestans can eventually lead to significant losses during storage.

Photograph: A potato field with advanced Phytophthora infestation.

Phytophthora thrives best under temperate temperatures and under relatively high humidity.

Quick penetration of the leave requires a sufficiently long wet leaf period. The disease

especially spreads via asexual spores (zoospores) which are formed by the white fluffy mould.

The disease is highly contagious. The spores are dispersed via wind or splashing rain drops.

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Photograph: The result of Phytophthora in affected tubers.

Phytophthora infestans survives winter as hyphae in affected tubers. Potato waste heaps are

notorious hideouts for the disease. Volunteer plants from tubers left in the field are also an

important way in which the disease can survive winter. Since a number of years, however, the

disease can also survive via sexual spores (oospores), which result from the joining of the so-

called A1 and A2 mating types. Earlier, the A2 mating type did not occur in the Belgian area

used for potato cultivation, so that survival via oospores is a relatively recent event. Oospores

survive up to 1 to 3 years in the soil and may cause the disease to spread, especially when crop

rotation turnover is too fast.

http://www.agris.be/nl/aardappelziekte/194003.asp#alg

Over the years, Phytophthora infestans turned more aggressive

Controlling the disease became more difficult over the years, as more aggressive isolates were

naturally selected within the Phytophthora infestans population and diversity has increased due

to recombination among various mating types previously not occurring within the same area.

The increasing aggressiveness is expressed in a shorter penetration time and the disease's life

cycle, more generations in a single growth season and a larger temperature tolerance.

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Phytophthora infestans causes huge costs

Close to 2 million hectares of potatoes are grown in Europe, and the harvest represents a value

of about 6 billion euros. According to estimates, late blight causes 1 billion euros of damage in

Europe. This damage consists of costs for controlling the disease (costs for purchasing

fungicides, plus costs for spray operations) and the costs that result from reductions in harvest

and losses during storage. To Belgian potato farmers the late blight means an annual cost of

roughly 55 million euros.

Haverkort et al., Societal Costs of Late Blight in Potato and Prospects of Durable Resistance Through Cisgenic

Modification, Potato Research (2008) 51:47-57

Environmental pressure caused by controlling Phytophthora infestans

In Belgium, large quantities of fungicide are used annually for ensuring sufficient control of

Phytophthora: over 1000 tons of active agent a year. In Flanders, an average of about 17 kg of

active substance is applied per hectare per year for controlling Phytophthora. The three main

active substances are (in order of decreasing importance): mancozeb, cymoxanil, and

propamocarb.

Source: Flemish Government, Department of Agriculture and Food, Section Monitoring and Analysis – LMN, data

2008

Ways to keep the disease pressure of Phytophthora low

Important measures of reducing the disease pressure are controlling volunteer plants and

destruction of potatoes in waste heaps. In some countries requirements exist to this purpose.

Doing this prevents Phytophthora pockets from further spreading the disease, but it does not

help in controlling sexual spores (oospores). Crop rotation that is sufficiently slow is effective

against spreading the disease via oospores. A second way to reduce disease pressure is by

carefully following the instructions of the monitoring services. A last resort is reducing potato

cultivation itself – the less potato production, the lower the disease pressure, but of course that

is hard to achieve in a country that is famous for its consumption of chips.

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Growing resistant varieties is the best way to reduce disease pressure

In a country with intensive potato production the best way to reduce disease pressure is

growing resistant varieties. The larger the area of cultivated resistant varieties, the lower the

disease pressure. In doing this, it is important to insert a sufficient number of different

resistance genes. If not, selection pressure will get too high or the limited amount of resistance

genes will lead to a quick adaptation of the Phytophthora population.

The area of cultivated resistant varieties probably does not need to be 100% to sufficiently

reduce the disease pressure. For comparison, look at the example of the virus resistant papaya

on Hawaii. Eighty percent of the cultivated area of papaya on this island is planted with

resistant crops, and that sufficiently reduces the disease pressure to be able to plant the

remaining 20% of the cultivated area with non-resistant varieties without leading to problems

with disease. Today, however, only a few potato varieties that are truly resistant are available:

The varieties Bionica, Toluca and Sarpo Mira. More about these varieties later.

Acting fast is essential when disease occurs

Acting fast becomes essential when Phytophthora is found in a field. In practice, the disease is

controlled by applying fungicides. Preventive action (treatment before penetration by the

mould of the plant takes place) is needed to get adequate results. Only a limited number of

agents have a sufficiently effective result.

Destruction of potato leafs and stems is another efficient method to prevent the disease from

spreading outside the field. However, leaf and stem destruction at a time when the crop is not

yet ready for the harvest has significant consequences. Certainly in late maturing varieties

strong tuber growth occurs in the last weeks before harvesting. Prematurely destructing the

potato leafs and stems would then result in lost yield potential. Instead of harvesting 45 to 60

tonnes per hectare perhaps only 35 tonnes are brought in, or even less. For this reason,

destruction is usually done on leafs and stems that are already dying.

Improved resistance with conventional plant breeding techniques

Bionica and Toluca

The Dutch have started over 40 years ago to crossbreed natural resistance genes into

commercially useful potato varieties. The resistance genes come from wild, tuber-forming

Solanaceae from the Andes. The very first crossbreeding took place in 1959. It took until 2005

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before some useful varieties were retrieved: Bionica and Toluca. Both varieties contain the

same, single resistance gene Rpi-blb-2 from Solanum bulbocastanum.

Crossing in resistance genes to the cultivated potato in a traditional way takes lots of work and

patience. This is due to the fact that the potato can not always be crossbred directly with wild

relatives in which the resistance genes are present. This necessitates application of a more

complicated crossing diagram, using so-called 'bridge cross breeding’. It only allows crossing in

one resistance gene at a time, unless you have the luck that the wild variant contains several

resistance genes and that at the same time those resistance genes are very close together in

the DNA of that wild variant. Furthermore, after crossbreeding with wild variants, many back-

crossings are needed to arrive back at the desired cultivated potato properties. Below the

crossing diagram is represented that led to the resistant variants Bionica and Toluca.

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www.meijer-potato.com

www.agrico.com

Sarpo Mira

Besides Bionca and Toluca there currently is a third potato variety on the market which features

high resistance: the Sarpo Mira variety. The origin of this variety is not to be found in The

Netherlands, but in Hungary. Like Bionica and Toluca, the variety has a long developmental

history. According to legend, reaching its current level of resistance took 40 years. In achieving

this, the following method was followed. As many different forms of resistance, tolerance and

oversensitivity as possible were collected. On the basis of this gene pool, crossbreeding was

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done as much as possible. The plants that resulted from the generated seeds were

subsequently sprayed with a mix of Phytophthora infestans that is as inclusive as possible.

When 95% of the plans have died, the Phytophthora in the remaining plants was neutralized

with a fungicide and these plants could reach maturity. The resulting tubers were subsequently

used for further work. This lasted until the collapse of the communist regimes. The institute

where the potatoes were kept was closed down. Thanks to the scientists involved, the best

material was saved.

The material resurfaced a number of years later, when a total contamination with Phytophthora

took place in Rumania. The Danish company Danespo then took over this material and provided

the Eastern European scientists involved with a new basis for continuing their work. From the

material that was subsequently obtained Sarpo Mira was marketed by Danespo.

www.danespo.com

Phytophthora easily overcomes single resistance genes

It is known that Phytophthora easily overcomes single resistances. The rich genetic diversity

within Phytophthora infestans populations implies that genotypes are already present or

emerge (the so-called mutants) and that these can circumvent the mechanism of the single

resistance gene that is present. This allows these mutants to have a selective advantage vis-à-

vis the wild types and subsequently quickly grow in numbers, dominating the population after a

short while. When several resistance genes are present in a potato variety, it is much more

difficult for the mould to adjust itself to both resistance genes at the same time. As stated

earlier, the varieties Bionica and Toluca contain the single resistance gene Rpi-blb2. This

resistance has not been overcome in the field for the moment. The varieties still offer adequate

protection. The fact that the varieties are cultivated on a small scale only – mainly by organic

and non-commercial vegetable gardeners – means that the selection pressure to overcome the

resistance is still limited. However, it is known that isolates exist on which the Rpi-blb2 gene has

no effect. That is why in The Netherlands the growers of varieties Bionica and Toluca are

explicitly asked to keep a close eye on their crop and immediately destruct the leafs and stems

if they observe any sign indicating the presence of Phytophthora. If they would fail to do so,

they could allow large-scale spreading of Phytophthora isolates that have overcome resistance.

And that would render 40 years of plant breeding useless in a relatively short period of time.

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Introducing resistance against Phytophthora infestans by means of genetic

manipulation

Since a number of years resistance genes are also introduced into potatoes by means of genetic

modification. This takes place in the U.S. (Cornell University), the U.K. (Sainsbury Laboratory,

John Innes Centre), Peru (Centro Internacional de la Papa (CIP)), Germany (BASF) and The

Netherlands (Wageningen UR), but not in Flanders. The advantage of the technology of genetic

modification vis-à-vis classical plant breeding is that one can develop resistant varieties much

faster and targeted and can also insert several resistance genes at the same time. Furthermore,

this insertion via genetic modification can be done without losing properties of the variety.

Genetic modification for resistance against Phytophthora infestans step-wise

STEP 1: Identifying resistance genes

For making potatoes resistant to Phytophthora, the resistance genes should be obtained

first. To do this, wild relatives of our cultivated potato are sown and tested for their

resistance. This is initially done with a simple in vitro test. This way, thousands of

different wild genotypes have been tested. Also, a so-called DNA fingerprint is made for

each of those plants. Next, a leaf test with a number of specific Phytophthora isolates is

performed on the plants that demonstrate resistance; this is to understand more about

the interaction with the mould. The plants that demonstrate resistance in these tests as

well are then subjected to field tests. In this way a smaller number of resistant

genotypes remain.

By comparing the DNA of the resistant plants with the plants that are not resistant, the

areas in that DNA can be located where the resistance is supposed to lay. Nowadays,

the further quest for resistance genes is made easier by the fact that the potato genome

at the base pair level is fully sequenced: the DNA order is known – every letter of it.

Every year, several new resistance genes are discovered. Today, about two dozen

different resistance genes are known and available. The isolated genes are also known

as cisgenes because they come from varieties that can be crossbred, which can also be

used for classical plant breeding.

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STEP 2: Modification of potato lines

Once you have a resistance gene, it can be inserted into the potato. This procedure

works as follows: as a first step, the gene is inserted into the DNA of soil bacterium

Agrobacterium tumefaciens. It is built into a piece of the bacterium’s DNA that is

naturally transmitted to plant DNA if the bacterium infects a plant.

Small pieces of potato tissue are brought onto a Petri dish in which a growing medium is

present in which the plant can grow. Next, the modified Agrobacterium is added to this

potato tissue. The bacterium infects the tissue and transmits the resistance genes to the

plant’s DNA. Then, a new plant can be generated from the potato tissue. This can be

done by adding various plant hormones to the growing medium, inducing the tissue to

develop roots and shoots.

Not all cells are infected by Agrobacterium, so the desired DNA fragment is not present

in all plant tissue cells. Therefore, plants should be selected that have actually

incorporated the DNA. This can be done in two ways. The first way is to couple another

gene to the resistance gene: a so-called selection marker. This can be an antibiotics

resistance gene, for example, or a gene providing herbicide tolerance. By then adding

the antibiotic or herbicide to the growing medium, only the plants survive that actually

incorporated the DNA. This is a simple way of selection.

The selection markers are often obtained from bacteria or from plants with which the

potato cannot naturally crossbreed. In other words, these are heterologous. Plants, in

which heterologous genes are inserted via genetic modification, are also known as

‘transgenic’.

In the second method of selection no selection marker is added to the DNA that is to be

transmitted. Al regenerated plants are allowed to grow up, including the ones that did

not incorporate DNA. Next, DNA is obtained from all developed plants. A genetic test

based on PCR1 determines whether a particular plant has incorporated the desired DNA.

This selection method is a lot more complicated and time-consuming. Plants that have

not incorporated the DNA are disposed of.

The resistant plants with selection markers, only contain DNA that is inserted via genetic

modification from potatoes or plants with which the potato can naturally crossbreed.

Such plants are also known as ‘cisgenic’. Cisgenic plants to a high degree are comparable

with plants which can be obtained via classical plant breeding. The only difference is the

technology with which the DNA is introduced. 1 PCR = Polymerase Chain Reaction

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STEP 3: Testing the plants in a greenhouse

The first growth stages of the genetically modified potatoes usually take place in a plant

nursery room. When the plants have become a bit bigger, they are brought to a

greenhouse, where they can continue growth in a pot or in the soil. Growing them in the

greenhouse has various purposes. Firstly, it is mainly about establishing whether the

desired properties are present. For this, Phytophthora resistance is the dominant

important property. Work only continues with plants that demonstrate resistance under

a simple leaf test. Also, variety characteristics are checked. Are all relevant properties

still there? Variety properties may be lost as a result of any of the next three

phenomena: (1) ‘insertional mutagenesis’, (2) ‘pleiotrophy’, of (3) ‘somaclonal

variation’.

Insertional mutagenesis indicates a mistake resulting from insertion of the additional

piece of DNA, which ends up in an existing gene, disrupting the function of this

particular gene. The potato is tetraploid, which means that it contains 4 copies of each

chromosome (4 ‘homologous chromosomes’). The inserted DNA ends up in one of those

four chromosomes, which means that even if the modification leads to a mutation, this

will involve only one of the four genes present in the four homologous chromosomes.

So the other three genes remain intact. Therefore, any mutation resulting from insertion

of DNA will almost never lead to a noticeable change in potato properties.

Pleiotrophy is the phenomenon which involves unexpected and undesirable effects

resulting from the fact that the additional piece of DNA ends up in an unexpected place

of the plant's DNA. This might for example disrupt the functioning of nearby genes.

Somaclonal variation is the emergence of phenotypic (= observable) deviations from the

potato from which it was derived, as a result of growing the plant in vitro and newly

regenerating the potato. This latter phenomenon is not specific for the technology of

genetic modification and also occurs during regular in vitro reproduction.

Therefore, work in the greenhouse requires strict selection, only selecting those plants

that are true-to-type, in other words, which still have all relevant variety properties. The

plants displaying resistance AND which are true-to-type are used for further work. With

this material, leaf tests are performed with real Phytophthora isolates. And plants that

also do well in these tests can in principle qualify for field tests.

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Cultivation of conventional resistant varieties

Potato varieties differ in their susceptibility for Phytophthora. Varieties such as Bintje and

Nicola are highly susceptible to the disease. Early varieties may potentially escape the biggest

part of the Phytophthora pressure due to their short growth cycle. Only a small number of

varieties such as Bionica, Toluca and Sarpo Mira contain true resistance based on one or several

R genes. The resistance genes coming from Solanum demissum that are present in some

varieties have often been overcome and therefore cannot be used anymore. Bionica, Toluca

and Sarpo Mira are marketed by Dutch firms Meijer and Agrico and Danish firm Danespo,

respectively. Bionica and Toluca are only cultivated for the market for fresh potatoes. Bionica

and Toluca potatoes are mainly used for organic farming. The potatoes are on the market since

a number of years and in 2009 their field resistance still turned out to be good. In Belgium,

Bionica is sold to non-commercial vegetable gardeners via AVEVE. In 2010 AVEVE sold seed

potatoes to non-commercial growers, translating to over 10 hectares of Bionica cultivation. Of

course this is only a drop in the ocean, but non-commercial cultivation is traditionally seen as a

potential source of spreading Phytophthora to commercial cultivation, because Phytophthora is

hardly controlled in non-commercial cultivation. In this way, a very small number of Bionica can

still have an impact on disease pressure.

Sarpo Mira still is not greatly appreciated by consumers due to its white/yellowish colour on the

inside, the flowery cooking result and its not-so-smooth tuber face (faint red skin, irregular

shape). The potato however turned out to be well suited for chips and flakes, granulates and

mush. McCain exclusively uses Sarpo Mira for their organic chips.

www.avevewinkels.be

Sustainable resistance

Single resistances against Phytophthora are unsustainable. Phytophthora will overcome such a

resistance sooner or later, and when that happens, the varieties with such single resistances

lose a great deal of their value. Combination of two different resistance genes offers a higher

degree of sustainability. This makes it not twice as difficult, but many times as difficult for

Phytophthora to overcome the resistance. Triple resistance will provide sustainability that is still

many times higher.

The most sustainable resistance is obtained if potato varieties with different multiple

resistances are alternated in space and time. Scientists are convinced that in such a scenario –

supplemented with some spraying – problems with Phytophthora should be a thing of the past,

as Phytophthora would not stand a chance of adapting itself.

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BASF’s Fortuna potato

BASF is most advanced in developing genetically modified, Phytophthora-resistant potatoes.

They have created the ‘Fortuna’ potato. This potato is close to its market introduction. The

potato contains two resistance genes, being Rpi-blb1, and Rpi-blb2. The combination of these

two resistance genes implies that the resistance of this potato should be much more

sustainable than that of the varieties Bionica and Toluca. The Fortuna potato also contains a

selection marker in the form of a mutated AHAS gene. This mutated AHAS gene provides

tolerance against sulfonyl-urea based herbicides, among others. BASF licensed the two

resistance genes from a company having the original property rights to the use of these genes.

BASF does not make any official statements about this, but according to various sources, the

Fortuna potato could have been derived from the Agri variety, a variety that is cultivated large-

scale in many places in Europe, and is equipped with the right properties for processing to

chips.

http://www.basf.com/group/corporate/en_GB/function/conversions:/publish/content/products-and-

industries/biotechnology/images/Fortuna_VC.pdf

The DuRPh cisgenic project

The DuRPh project started in The Netherlands in 2006. DuRPh stands for ‘Duurzame Resistentie

tegen Phytophthora’ or Sustainable Resistance against Phytophthora. This is a 10-year project

financed by the Dutch government which aims at identifying and characterising resistance

genes, creating and testing genetically modified potato lines, and communicate this to a large

target audience. The ultimate goal is to present a proof of principle with regard to sustainable

resistance against this disease. The project was set up from a genuine sustainability approach

and takes into account economic aspects, as well as ecological and social aspects. Wageningen

UR took the initiative for the DuRPh project and carries it out.

In the DuRPh project, genetically modified potatoes are developed with a specific underlying

idea. Of course the technology of genetic modification is used, and the end result is still subject

to GMO legislation, but the plants that are created have the highest possible degree of

similarity with plants that can also be obtained via conventional plant breeding. This is called

cisgenesis, which implies that:

(1) The resistance genes come from Solanaceae with which the potato can naturally

crossbreed.

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(2) The resistance genes are not tampered with. They are inserted with their natural

regulation signals.

(3) No selection markers will be available in the final lines, only cisgenes.

Work with constructs in which a marker is present is used in the first phases of development

only, just because this is easier and takes less time, and a quicker insight is obtained in which

genes (gene combinations) provide adequate and sustainable resistance.

Since 2006, Wageningen UR performs small-scale field tests with genetically modified

Phytophthora-resistant potato lines on various locations in The Netherlands. In these field tests,

the various lines that display adequate resistance during greenhouse testing are subjected to a

first reality check. In the course of years, various constructs and lines are tested in this way.

There are potatoes that include single resistance, double resistance, triple resistance, and lines

with (transgenic) and without selection marker (cisgenic).

www.durph.nl

Cisgenesis is subject of regulatory discussions in the EU

Cisgenesis is genetic modification; there is no question about that. However, there is a

discussion about the question whether cisgenesis should stay subject to GMO regulation or that

it might be exempt from it. The existing legislation already provides a number of exceptions.

Classical mutagenesis, for example, falls under the definition of genetic modification, but is

exempt from European GMO legislation. A technical working group now considers the question

whether there are any sound reasons to keep cisgenesis under GMO legislation or not. The

working group does not only scrutinises cisgenesis, but also looks at a number of other modern

ways to achieve DNA modifications, among which are reverse breeding and targeted DNA

repair.

Genetically modified Phytophthora-resistant potatoes do not threaten the

environment

Genetically modified plants should not have negative effects on the environment, such as

pushing out existing species in nature or disturbing delicate natural balances. Genetically

modified Phytophthora-resistant potatoes include resistance genes that naturally occur in wild,

tuber-forming Solanaceae. The same or comparable genes are also present in Phytophthora-

resistant potatoes that are obtained via conventional plant breeding. Therefore, the genes in

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the genetically modified potato lines are just as safe to the environment as the same genes in

the varieties obtained via conventional plant breeding and those in wild relatives. Is that the

end of the story? Not quite. It cannot be completely ruled out that genetic modification leads to

unexpected phenomena having an effect on the survival of the potato plant in nature, or that

other unexpected changes in plant properties emerge. For this reason, strict selection takes

place during the quest for suitable genetically modified lines, in order to only select those lines

that kept the right variant properties. In field tests, the phenotype and behaviour of the plants

also undergo exhaustive scrutiny in order to locate any deviations, and to look whether

deviations have undesirable consequences. Plants with undesirable properties will not be

marketed.

In Europe, the potato is absent as a wild plant. Furthermore, the potato has no relatives with

which it could crossbreed. Black nightshade is phylogenetically closest to the potato, but cannot

be interbred with it. Furthermore, the cultivated potato is a highly domesticated plant that will

not suddenly colonise nature. The plant lacks the power to successfully compete with wild

plants. In practice, this means that neither the genetically modified potatoes, nor the

genetically modified property itself will be able to colonise our environment. The potato will

only be able to appear as volunteer plants in fields where it has previously grown, and in soil

coming from such fields. These cases will, however, always be a temporary affair.

Food safety of genetically modified Phytophthora-resistant potatoes

Genetically modified Phytophthora-resistant potatoes contain the same or comparable

resistance genes as traditional Phytophthora-resistant potatoes do. To provide a practical

example: BASF’s genetically modified Phytophthora potato and the potato varieties Bionica and

Toluca that have been obtained via conventional plant breeding, all contain the same Rpi-blb2

gene. For the genetically modified potatoes, the safety of the gene product that is synthesized

by the resistance gene should be demonstrated. It should be demonstrated that the product is

not toxic and does not cause allergic reactions. This is not a requirement in varieties like Bionica

or Toluca which are the result of conventional plant breeding. These varieties can be marketed

without further food safety tests. And all this despite the fact that the Rpi-blb2 gene in these

plants is surrounded by hundreds of genes originating from the wild variety.

As indicated earlier, it cannot be ruled out that genetic modification can lead to unexpected

side effects. Insertional mutagenesis, pleiotrophy and somaclonal variation were mentioned as

possible causes of such side effects. For this reason, a genetically modified Phytophthora-

resistant potato should undergo additional tests. The composition of the potato should be

compared with that of conventional potatoes, for example. For this purpose the quantity of the

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main components of the potato is measured and compared. The OECD determined which

substances should be included in the analysis. The potato naturally contains toxic

glycoalkaloids. Their presence is the reason why one should peel potatoes before they can be

safely eaten. Besides, the potato contains substances blocking our digestive enzymes. Eating

raw potatoes leads to stomach-ache, so to prevent this we cook them before consumption. In

the required analysis, the quantity of these kinds of substances is closely watched, because

everything should be done to prevent marketing potatoes that carry too many hazardous

substances. By the way, the amount of glycoalkaloids is checked for every new variety before

market entry, whether it is genetically modified or not. In The Netherlands and Sweden an

upper safety limit of a total glycoalkaloid level of 200 mg/kg applies. Other EU countries have

not specified glycoalkaloid limits.

If analysis of the composition of the genetically modified potato shows an unexpected deviation

in the level of certain substances, additional tests should be performed. Usually this takes the

form of a 90-day food test on rats.

OECD, Consensus Document on the Biology of Solanum tuberosum subspecies tuberosum (potato)

Genetically modified Phytophthora-resistant potatoes not on the market for

now

BASF's genetically modified Phytophthora-resistant Fortuna potato reached an advanced stage,

but is still not on the market. BASF should first apply for market admission with the European

Commission to achieve this. And once that file has been submitted, it will take some years more

before the full procedure is completed. It is hard to look into the future, but it is expected to

last until 2013 before a genetically modified Phytophthora-resistant potato is available on the

market. All other lines that are being developed are less far advanced than the Fortuna potato

and their market introduction will take a lot more time.